Tissue engineering has become a key therapeutic tool in the treatment of damaged or diseased organs and tissues, such as blood vessels and urinary bladders. Tissue engineering seeks to provide regenerated tissue architectures in vitro, but has not yet successfully created thick, highly vascularized, multi-functional tissues replicating native structure. Cell sheet engineering has been proposed to overcome the problems of tissue engineering. Cell sheet engineering can avoid tissue reconstruction limitations using biodegradable scaffolds or single cell suspension injection. Such a cell sheet constructed in vitro could be useful in various clinical situations to regenerate tissues (especially epithelial tissues) such as artificial skin and artificial cornea.
Cell sheets can be prepared using cells from various biological tissues and organs. These can be used alone as tissue for transplantation or in combination with sheets composed of other cells, and can be used either singly or in stacked arrangements of multiple cell sheets. Cells normally attach strongly to hydrophobic surfaces but weakly to hydrophilic surfaces. When cells are cultured in a normal container such as culture dish, they proliferate and make a monolayer sheet. The cells are structurally connected to each other by intercellular junction proteins and adhere to the surface of the culture dish with adhesive proteins. In the art, in order to detach the cell sheet from the dish, proteolytic enzymes are normally used to disrupt the adhesive proteins. However, in such a releasing procedure using a proteolytic enzyme, not only may the cells be damaged, but also the extracellular matrix produced during the culture may be decomposed. Furthermore, since most proteolytic enzymes are materials derived from animals, the application of such proteolytic enzymes to a cell sheet for regenerative medicine is problematic in terms of safety. Therefore, it is desirable to recover the monolayer cells in a cell sheet structure at the end of the culture stage without using a biochemical or chemical reagent.
Subsequently, cell sheets are developed with thermo responsive culture dishes. Thermo responsive polymers are grafted to dishes covalently, which allows different cell types to attach and proliferate at 37° C. Cells detach spontaneously when temperature decreases to below 32° C., without the need to use enzymes, and this is due to the natural specification of the intelligent polymers also due to the detachment of the cell metabolic changes made by the polymer resulting from decreasing temperature. U.S. Pat. No. 6,956,077 discloses a polymer compound which undergoes stretching and cohesion with a change in the polarity of the polymer per se due to a temperature change, a process for producing this polymer compound, a heat-responsive polymer material containing this compound, a separation method with the use of a material containing this heat-responsive polymer material, and a method for separating chemicals, biological polymers (proteins, peptides, etc.) and biological samples (cells, etc.) by using this material. Hironobu Takahashi et al. demonstrated surface-initiated RAFT technique to materialize new type thermoresponsive N-isopropylarylamide (PIPAAm) brush surfaces for cell sheet harvest (Biomacromolecules, “Controlled Chain Length And Graft Density Of Thermoresponsive Polymer Brushes For Optimizing Cell Sheet Harvest” Vol. 11, No. 8, 2010, pp. 1991-1999). US 2006240552 A1 provides an anterior ocular segment related cell sheet or three-dimensional structure and a process for producing the anterior ocular segment related cell sheet or three-dimensional structure by using a temperature responsive polymer having an upper or lower critical dissolution temperature of 0-80° C. US 20080131476 A1 provides a cultured cell sheet with good tissue adherence and flexibility which is obtained by culturing cells on a support for cell culture in which a surface of a substrate is coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature of 0-80° C. along with a surfactant protein or a crosslinking inhibitor.
However, the above-mentioned prior references use thermoresponsive polymers grafted on the surface of a culture dish and control temperature to release the cultured cells. The monomers (such as PIPAAm) of the polymers may remain in the culture dish so as to bring toxicity and the temperature control procedure is not simple. Therefore, there is a need to develop a substrate with low toxicity and improved cell attachment and detachment and a process of simply detaching cell sheets from the cultured container.